Template plasmid integration in germline genome-edited cattle

Norris, Alexis L.; Lee, Stella S.; Greenlees, Kevin J.; Tadesse, Daniel A.; Miller, Mayumi F.; Lombardi, Heather A.

doi:10.1101/715482

Cited by 30 publications

(30 citation statements)

References 17 publications

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“…The main conclusion from our current results is that knock-in integration landscapes can be very heterogeneous and condition-dependent, and therefore need to be characterized more systematically. The recent debate surrounding the discovery of plasmid backbone sequences in the genome of engineered cattle 23,24 (derived from mis-integration of an HDR donor template) also highlights the universal need for comprehensive genotyping post-editing. Most existing analytical procedures seek to assess editing conditions based on a limited set of heuristic variables (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, cells edited using plasmid HDR donor displayed a strong signature of integration of plasmid backbone sequence past homology arm boundaries ( Figures 1G and S1H). Interestingly, high rates of backbone integration from plasmid donors have been previously reported in a systematic study of gene editing in human induced pluripotent stem cells (iPSCs) 7 , and in the genome of cattle engineered using plasmid HDR templates 23,24 . Of note, knock-knock can also quantify a variety of additional editing outcomes including spurious integration of exogenous DNA sequences (such as rare integrations of E. coli genomic DNA presumably carried over from plasmid preparation, Figure S1G) or longer range deletions 25 which often arise at micro-homology overlaps ( Figure S1I).…”

Section: Introductionmentioning

confidence: 99%

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Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

Canaj

Hussmann

et al. 2019

Preprint

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CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knockin of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on longread sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.Recent developments in gene editing technologies have transformed our ability to manipulate genomes. Programmable site-specific nucleases -in particular CRISPR/Cas systems -introduce double-strand breaks (DSBs) at chosen genomic locations, prompting the activation of two separate DNA repair pathways which can be leveraged for genome engineering 1 . On the one hand, nonhomologous end-joining (NHEJ) can introduce insertions or deletions (in-dels) at the DSB site to inactivate gene products or regulatory elements. On the other hand, homology-directed repair (HDR)can use exogenous DNA sequences as templates to integrate (knock-in) new genetic information in the locus of interest 2 . Knock-in strategies have wide applications ranging from correcting diseasecausing mutations or inserting therapeutic payloads in a clinical context 3-5 , to introducing functional reporters for cell biology research 6,7 .Ultimately, the power of genetic engineering will rely on our ability to predictably edit genomes in order to precisely control cellular behaviors. Traditionally, NHEJ and HDR have been set apart by their degree of predictability: NHEJ is often thought to drive random repair outcomes, while HDR is considered to enable precise and templated editing. Recent data is challenging this simple distinction, however. High-throughput sequencing of NHEJ repair has uncovered complex but actionable sequence patterns that control in-del outcomes at DSBs 8-13 . In some cases, predictable NHEJ outcomes can be leveraged to precisely correct pathogenic human mutations, paving the way for template-free therapeutic genome editing 9 . By contrast, while accumulating evidence suggests that the integration of payload in knock-in experiments is not always precise 7,14,15 , a systematic and quantitative profiling of the full spectrum of HDR repair outcomes is still missing.Given its wide range of applications in both clinical and research settings, understanding the parameters that govern the efficiency and precision of knock...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

Canaj

Hussmann

et al. 2019

Preprint

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“…It allows direct insertion of a premature termination codon, which prevents the formation of novel protein sequences with unknown function after NEHJ 30 . However, caution is required when selecting strains to ensure the expression plasmid has not integrated into the genome, which has been shown to happen with the backbone of HDR constructs for introducing the hornlessness (polled) trait in cattle 31,32 . Plasmid integration may have been favoured by the transient puromycin selection we applied to enhance editing efficiency.…”

Section: Discussionmentioning

confidence: 99%

Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

McLean

Appleby

Wei

et al. 2019

Preprint

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Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem and produce chimaeras with absolute donor germline transmission, we have disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals.Using Cas9 mediated homology-directed repair (HDR), we introduced a DAZL loss-offunction mutation in male ovine fetal fibroblasts. Following manual single-cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL-null male neonatal sheep lacked germ cells. Somatic cells within their testes were morphologically intact and expressed normal levels of somatic cell-specific marker genes, indicating that the germ cell niche remained intact. This extends the DAZL-mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute transmitters in rapid livestock improvement.

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“…In a similar fashion, thermotolerant Angus cattle were obtained by introduction of the SLICK hair locus, responsible for less dense hair and increased thermal transpiration [30,43] . It is important to note that recent analysis of the genome of the hornless cattle developed by Carlson et al revealed, in addition to the desired introduction of the P c POLLED locus, an unintended duplication of P c POLLED and chromosomal integration of the full-length repair template plasmid backbone [44,45] . These unintended plasmid integrations were also inherited by offspring of the gene-edited bulls [45] .…”

Section: Developments In Genetic Modification Of Cattlementioning

confidence: 99%

Developments in genetic modification of cattle and implications for regulation, safety and traceability

Berg¹,

Kleter²,

Battaglia³

et al. 2020

Front. Agr. Sci. Eng.

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Genetic modification techniques, in particular novel gene editing technologies, hold the yet unfulfilled promise of altering genetic traits in farm animals more efficiently than by crossbreeding, allowing for a more rapid development of new cattle breeds with distinct traits. Gene editing technologies allow for the directed alteration of specific traits and thereby have the potential to enhance, for instance, disease resilience, production yield and the production of desired substances in milk. The potential implications of these technological advancements, which are often combined with animal cloning methods, are discussed both for animal health and for consumer safety, also with consideration of available methods for the detection and identification of the related products in the food supply chain. Finally, an overview is provided of current regulatory approaches in the European Union (EU) and major countries exporting beef to the EU, for products from animals bred through established practices as well as modern biotechnologies.

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Template plasmid integration in germline genome-edited cattle

Abstract: 19We analyzed publicly available whole genome sequencing data from cattle which were 20 germline genome-edited to introduce polledness. Our analysis discovered the unintended 21 heterozygous integration of the plasmid and a second copy of the repair template sequence, 22

Cited by 30 publications

References 17 publications

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

Developments in genetic modification of cattle and implications for regulation, safety and traceability

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